Accelerating drug discovery and repurposing by combining transcriptional signature connectivity with docking.

We present an in silico approach for drug discovery, dubbed connectivity enhanced structure activity relationship (ceSAR). Building on the landmark LINCS library of transcriptional signatures of drug-like molecules and gene knockdowns, ceSAR combines cheminformatic techniques with signature concordance analysis to connect small molecules and their targets and further assess their biophysical compatibility using molecular docking. Candidate compounds are first ranked in a target structure-independent manner, using chemical similarity to LINCS analogs that exhibit transcriptomic concordance with a target gene knockdown. Top candidates are subsequently rescored using docking simulations and machine learning-based consensus of the two approaches. Using extensive benchmarking, we show that ceSAR greatly reduces false-positive rates, while cutting run times by multiple orders of magnitude and further democratizing drug discovery pipelines. We further demonstrate the utility of ceSAR by identifying and experimentally validating inhibitors of BCL2A1, an important antiapoptotic target in melanoma and preterm birth-associated inflammation.

Blood immunophenotyping identifies distinct kidney histopathology and outcomes in patients with lupus nephritis.

Lupus nephritis (LN) is a frequent manifestation of systemic lupus erythematosus, and fewer than half of patients achieve complete renal response with standard immunosuppressants. Identifying non-invasive, blood-based pathologic immune alterations associated with renal injury could aid therapeutic decisions. Here, we used mass cytometry immunophenotyping of peripheral blood mononuclear cells in 145 patients with biopsy-proven LN and 40 healthy controls to evaluate the heterogeneity of immune activation in patients with LN and to identify correlates of renal parameters and treatment response. Unbiased analysis identified 3 immunologically distinct groups of patients with LN that were associated with different patterns of histopathology, renal cell infiltrates, urine proteomic profiles, and treatment response at one year. Patients with enriched circulating granzyme B+ T cells at baseline showed more severe disease and increased numbers of activated CD8 T cells in the kidney, yet they had the highest likelihood of treatment response. A second group characterized primarily by a high type I interferon signature had a lower likelihood of response to therapy, while a third group appeared immunologically inactive by immunophenotyping at enrollment but with chronic renal injuries. Main immune profiles could be distilled down to 5 simple cytometric parameters that recapitulate several of the associations, highlighting the potential for blood immune profiling to translate to clinically useful non-invasive metrics to assess immune-mediated disease in LN.

The Proteasome Inhibitor Bortezomib Induces p53-Dependent Apoptosis in Activated B Cells.

The proteasome inhibitor bortezomib (BTZ) is proposed to deplete activated B cells and plasma cells. However, a complete picture of the mechanisms underlying BTZ-induced apoptosis in B lineage cells remains to be established. In this study, using a direct in vitro approach, we show that deletion of the tumor suppressor and cell cycle regulator p53 rescues recently activated mouse B cells from BTZ-induced apoptosis. Furthermore, BTZ treatment elevated intracellular p53 levels, and p53 deletion constrained apoptosis, as recently stimulated cells first transitioned from the G1 to S phase of the cell cycle. Moreover, combined inhibition of the p53-associated cell cycle regulators and E3 ligases MDM2 and anaphase-promoting complex/cyclosome induced cell death in postdivision B cells. Our results reveal that efficient cell cycle progression of activated B cells requires proteasome-driven inhibition of p53. Consequently, BTZ-mediated interference of proteostasis unleashes a p53-dependent cell cycle-associated death mechanism in recently activated B cells.

Effects of invivo CXCR4 blockade and proteasome inhibition on bone marrow plasma cells in HLA-sensitized kidney transplant candidates.

To date, plasma cell (PC)-targeted therapies have been limited by suboptimal PC depletion and antibody rebound. We hypothesized this is partly because of PC residence in protective bone marrow (BM) microenvironments. The purpose of this proof-of-concept study was to examine the effects of the CXCR4 antagonist, plerixafor, on PC BM residence; its safety profile (alone and in combination with a proteasome inhibitor, bortezomib); and the transcriptional effect on BMPCs in HLA-sensitized kidney transplant candidates. Participants were enrolled into 3 groups: group A (n = 4), plerixafor monotherapy; and groups B (n = 4) and C (n = 4), plerixafor and bortezomib combinations. CD34+ stem cell and PC levels increased in the blood after plerixafor treatment. PC recovery from BM aspirates varied depending on the dose of plerixafor and bortezomib. Single-cell RNA sequencing on BMPCs from 3 group C participants pretreatment and posttreatment revealed multiple populations of PCs, with a posttreatment enrichment of oxidative phosphorylation, proteasome assembly, cytoplasmic translation, and autophagy-related genes. Murine studies demonstrated dually inhibiting the proteasome and autophagy resulted in greater BMPC death than did monotherapies. In conclusion, this pilot study revealed anticipated effects of combined plerixafor and bortezomib on BMPCs, an acceptable safety profile, and suggests the potential for autophagy inhibitors in desensitization regimens.

An atlas of gene regulatory networks for memory CD4 + T cells in youth and old age.

Aging profoundly affects immune-system function, promoting susceptibility to pathogens, cancers and chronic inflammation. We previously identified a population of IL-10-producing, T follicular helper-like cells (" Tfh10 "), linked to suppressed vaccine responses in aged mice. Here, we integrate single-cell ( sc )RNA-seq, scATAC-seq and genome-scale modeling to characterize Tfh10 - and the full CD4 + memory T cell ( CD4 + TM ) compartment - in young and old mice. We identified 13 CD4 + TM populations, which we validated through cross-comparison to prior scRNA-seq studies. We built gene regulatory networks ( GRNs ) that predict transcription-factor control of gene expression in each T-cell population and how these circuits change with age. Through integration with pan-cell aging atlases, we identified intercellular-signaling networks driving age-dependent changes in CD4 + TM. Our atlas of finely resolved CD4 + TM subsets, GRNs and cell-cell communication networks is a comprehensive resource of predicted regulatory mechanisms operative in memory T cells, presenting new opportunities to improve immune responses in the elderly.

T-cell infiltrate intensity is associated with delayed response to treatment in late acute cellular rejection in pediatric liver transplant recipients.

BACKGROUND: Late acute cellular rejection (ACR) is associated with donor-specific antibodies (DSA) development, chronic rejection, and allograft loss. However, accurate predictors of late ACR treatment response are lacking. ACR is primarily T-cell mediated, yet B cells and plasma cells (PC) also infiltrate the portal areas during late ACR. To test the hypothesis that the inflammatory milieu is associated with delayed response (DR) to rejection therapy, we performed a single-center retrospective case-control study of pediatric late liver ACR using multiparameter immunofluorescence for CD4, CD8, CD68, CD20, and CD138 to identify immune cell subpopulations. METHODS: Pediatric liver transplant recipients transplanted at <17 years of age and treated for biopsy-proven late ACR between January 2014 and 2019 were stratified into rapid response (RR) and DR based on alanine aminotransferase (ALT) normalization within 30 days of diagnosis. All patients received IV methylprednisolone as an initial rejection treatment. Immunofluorescence was performed on archived formalin-fixed paraffin embedded (FFPE) liver biopsy tissue. RESULTS: Liver biopsies from 60 episodes of late ACR in 54 patients were included in the analysis, of which 33 were DR (55%). Anti-thymocyte globulin was only required in the DR group. The frequency of liver-infiltrating CD20+ and CD8+ lymphocytes and the prevalence of autoantibodies were higher in the DR group. In univariate logistic regression analysis, serum gamma-glutamyl transpeptidase (GGT) level at diagnosis, but not ALT, Banff score or presence of DSA, predicted DR. CONCLUSIONS: Higher serum GGT level, presence of autoantibodies, and increased CD8+ T-cell infiltration portends DR in late ACR treatment in children.

Farnesoid X receptor antagonizes macrophage-dependent licensing of effector T lymphocytes and progression of sclerosing cholangitis.

Immune-mediated bile duct epithelial injury and toxicity of retained hydrophobic bile acids drive disease progression in fibrosing cholangiopathies such as biliary atresia or primary sclerosing cholangitis. Emerging therapies include pharmacological agonists to farnesoid X receptor (FXR), the master regulator of hepatic synthesis, excretion, and intestinal reuptake of bile acids. Unraveling the mechanisms of action of pharmacological FXR agonists in the treatment of sclerosing cholangitis (SC), we found that intestinally restricted FXR activation effectively reduced bile acid pool size but did not improve the SC phenotype in MDR2-/- mice. In contrast, systemic FXR activation not only lowered bile acid synthesis but also suppressed proinflammatory cytokine production by liver-infiltrating inflammatory cells and blocked progression of hepatobiliary injury. The hepatoprotective activity was linked to suppressed production of IL1ß and TNFα by hepatic macrophages and inhibition of TH1/TH17 lymphocyte polarization. Deletion of FXR in myeloid cells caused aberrant TH1 and TH17 lymphocyte responses in diethoxycarbonyl-1,4-dihydrocollidine-induced SC and rendered these mice resistant to the anti-inflammatory and liver protective effects of systemic FXR agonist treatment. Pharmacological FXR activation reduced IL1ß and IFNγ production by liver- and blood-derived mononuclear cells from patients with fibrosing cholangiopathies. In conclusion, we demonstrate FXR to control the macrophage-TH1/17 axis, which is critically important for the progression of SC. Hepatic macrophages are cellular targets of systemic FXR agonist therapy for cholestatic liver disease.

Bcl-2 Is Necessary to Counteract Bim and Promote Survival of TCRαß+CD8αα+ Intraepithelial Lymphocyte Precursors in the Thymus.

The precursors of TCRαß+CD8αα+ intraepithelial lymphocytes (IEL) arise in the thymus through a complex process of agonist selection. We and others have shown that the proapoptotic protein, Bim, is critical to limit the number of thymic IEL precursors (IELp), as loss of Bim at the CD4+CD8+ double-positive stage of development drastically increases IELp. The factors determining this cell death versus survival decision remain largely unknown. In this study, we used CD4CreBcl2f/f mice to define the role of the antiapoptotic protein Bcl-2 and CD4CreBcl2f/fBimf/f mice to determine the role of Bcl-2 in opposing Bim to promote survival of IELp. First, in wild-type mice, we defined distinct subpopulations within PD-1+CD122+ IELp, based on their expression of Runx3 and α4ß7. Coexpression of α4ß7 and Runx3 marked IELp that were most dependent upon Bcl-2 for survival. Importantly, the additional loss of Bim restored Runx3+α4ß7+ IELp, showing that Bcl-2 antagonizes Bim to enable IELp survival. Further, the loss of thymic IELp in CD4CreBcl2f/f mice also led to a dramatic loss of IEL in the gut, and the additional loss of Bim restored gut IEL. The loss of gut IEL was due to both reduced seeding by IELp from the thymus as well as a requirement for Bcl-2 for peripheral IEL survival. Together, these findings highlight subset-specific and temporal roles for Bcl-2 in driving the survival of TCRαß+CD8αα+ IEL and thymic IELp.

PD1 blockade enhances K+ channel activity, Ca2+ signaling, and migratory ability in cytotoxic T lymphocytes of patients with head and neck cancer.

BACKGROUND: Immunotherapy has emerged as a promising treatment modality for head and neck squamous cell carcinoma (HNSCC). Pembrolizumab, an anti-programmed death 1 antibody, is an immunotherapy agent currently approved for metastatic HNSCC and curative intent clinical trials. Although clinical responses to pembrolizumab are promising, many patients fail to respond. However, it is well known that T cell cytotoxicity and chemotaxis are critically important in the elimination of HNSCC tumors. These functions depend on ion channel activity and downstream Ca2+ fluxing abilities, which are defective in patients with HNSCC. The purpose of this study was to elucidate the effects of pembrolizumab on potassium (K+) channel (KCa3.1 and Kv1.3) activity, Ca2+ fluxes, and chemotaxis in the cytotoxic T cells of patients with HNSCC and to determine their correlation with treatment response. METHODS: Functional studies were conducted in CD8+ peripheral blood T cells (PBTs) and tumor infiltrating lymphocytes (TILs) from patients with HNSCC treated with pembrolizumab. Untreated patients with HNSCC were used as controls. The ion channel activity of CD8+ T cells was measured by patch-clamp electrophysiology; single-cell Ca2+ fluxing abilities were measured by live microscopy. Chemotaxis experiments were conducted in a three-dimensional collagen matrix. Pembrolizumab patients were stratified as responders or non-responders based on pathological response (percent of viable tumor remaining at resection; responders: ≤80% viable tumor; non-responders: >80% viable tumor). RESULTS: Pembrolizumab increased K+ channel activity and Ca2+ fluxes in TILs independently of treatment response. However, in PBTs from responder patients there was an increased KCa3.1 activity immediately after pembrolizumab treatment that was accompanied by a characteristic increase in Kv1.3 and Ca2+ fluxes as compared with PBTs from non-responder patients. The effects on Kv1.3 and Ca2+ were prolonged and persisted after tumor resection. Chemotaxis was also improved in responder patients' PBTs. Unlike non-responders' PBTs, pembrolizumab increased their ability to chemotax in a tumor-like, adenosine-rich microenvironment immediately after treatment, and additionally they maintained an efficient chemotaxis after tumor resection. CONCLUSIONS: Pembrolizumab enhanced K+ channel activity, Ca2+ fluxes and chemotaxis of CD8+ T cells in patients with HNSCC, with a unique pattern of response in responder patients that is conducive to the heightened functionality of their cytotoxic T cells.

A prospective, iterative, adaptive trial of carfilzomib-based desensitization.

Proteasome inhibitor-based strategies hold promise in transplant but have yielded varying results. Carfilzomib, a second-generation proteasome inhibitor, may possess advantages over bortezomib, the first-generation proteasome inhibitors. The purpose of this study was to evaluate the safety, toxicity, and preliminary efficacy of carfilzomib in highly HLA-sensitized kidney transplant candidates. Renal transplant candidates received escalating doses of carfilzomib followed by plasmapheresis (group A) or an identical regimen with additional plasmapheresis once weekly before carfilzomib dosing. Thirteen participants received carfilzomib, which was well tolerated with most adverse events classified as low grade. The safety profile was similar to bortezomib desensitization; however, neurotoxicity was not observed with carfilzomib. Toxicity resulted in permanent dose reduction in 1 participant but caused no withdrawals or deaths. HLA antibodies were substantially reduced with carfilzomib alone, and median maximal immunodominant antibody reduction was 72.8% (69.8% for group A, P = .031, 80.1% for group B, P = .938). After depletion, rebound occurred rapidly and antibody levels returned to baseline between days 81 and 141. Bone marrow studies revealed that approximately 69.2% of plasma cells were depleted after carfilzomib monotherapy. Carfilzomib monotherapy-based desensitization provides an acceptable safety and toxicity profile while leading to significant bone marrow plasma cell depletion and anti-HLA antibody reduction.

mTOR Inhibitor Therapy Diminishes Circulating CD8+ CD28- Effector Memory T Cells and Improves Allograft Inflammation in Belatacept-refractory Renal Allograft Rejection.

BACKGROUND: Renal allograft rejection is more frequent under belatacept-based, compared with tacrolimus-based, immunosuppression. We studied kidney transplant recipients experiencing rejection under belatacept-based early corticosteroid withdrawal following T-cell-depleting induction in a recent randomized trial (Belatacept-based Early Steroid Withdrawal Trial, clinicaltrials.gov NCT01729494) to determine mechanisms of rejection and treatment. METHODS: Peripheral mononuclear cells, serum creatinine levels, and renal biopsies were collected from 8 patients undergoing belatacept-refractory rejection (BRR). We used flow cytometry, histology, and immunofluorescence to characterize CD8 effector memory T cell (TEM) populations in the periphery and graft before and after mammalian target of rapamycin (mTOR) inhibition. RESULTS: Here, we found that patients with BRR did not respond to standard antirejection therapy and had a substantial increase in alloreactive CD8 T cells with a CD28/DR/CD38/CD45RO TEM. These cells had increased activation of the mTOR pathway, as assessed by phosphorylated ribosomal protein S6 expression. Notably, everolimus (an mTOR inhibitor) treatment of patients with BRR halted the in vivo proliferation of TEM cells and their ex vivo alloreactivity and resulted in their significant reduction in the peripheral blood. The frequency of circulating FoxP3 regulatory T cells was not altered. Importantly, everolimus led to rapid resolution of rejection as confirmed by histology. CONCLUSIONS: Thus, while prior work has shown that concomitant belatacept + mTOR inhibitor therapy is effective for maintenance immunosuppression, our preliminary data suggest that everolimus may provide an available means for effecting "rescue" therapy for rejections occurring under belatacept that are refractory to traditional antirejection therapy with corticosteroids and polyclonal antilymphocyte globulin.

The immune cell landscape in kidneys of patients with lupus nephritis.

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.

IL-1 signaling mediates intrauterine inflammation and chorio-decidua neutrophil recruitment and activation.

Neutrophil infiltration of the chorioamnion-decidua tissue at the maternal-fetal interface (chorioamnionitis) is a leading cause of prematurity, fetal inflammation, and perinatal mortality. We induced chorioamnionitis in preterm rhesus macaques by intraamniotic injection of LPS. Here, we show that, during chorioamnionitis, the amnion upregulated phospho-IRAK1-expressed neutrophil chemoattractants CXCL8 and CSF3 in an IL-1-dependent manner. IL-1R blockade decreased chorio-decidua neutrophil accumulation, neutrophil activation, and IL-6 and prostaglandin E2 concentrations in the amniotic fluid. Neutrophils accumulating in the chorio-decidua had increased survival mediated by BCL2A1, and IL-1R blockade also decreased BCL2A1+ chorio-decidua neutrophils. Readouts for inflammation in a cohort of women with preterm delivery and chorioamnionitis were similar to findings in the rhesus macaques. IL-1 is a potential therapeutic target for chorioamnionitis and associated morbidities.

Manipulating DNA damage-response signaling for the treatment of immune-mediated diseases.

Antigen-activated lymphocytes undergo extraordinarily rapid cell division in the course of immune responses. We hypothesized that this unique aspect of lymphocyte biology leads to unusual genomic stress in recently antigen-activated lymphocytes and that targeted manipulation of DNA damage-response (DDR) signaling pathways would allow for selective therapeutic targeting of pathological T cells in disease contexts. Consistent with these hypotheses, we found that activated mouse and human T cells display a pronounced DDR in vitro and in vivo. Upon screening a variety of small-molecule compounds, we found that potentiation of p53 (via inhibition of MDM2) or impairment of cell cycle checkpoints (via inhibition of CHK1/2 or WEE1) led to the selective elimination of activated, pathological T cells in vivo. The combination of these strategies [which we termed "p53 potentiation with checkpoint abrogation" (PPCA)] displayed therapeutic benefits in preclinical disease models of hemophagocytic lymphohistiocytosis and multiple sclerosis, which are driven by foreign antigens or self-antigens, respectively. PPCA therapy targeted pathological T cells but did not compromise naive, regulatory, or quiescent memory T-cell pools, and had a modest nonimmune toxicity profile. Thus, PPCA is a therapeutic modality for selective, antigen-specific immune modulation with significant translational potential for diverse immune-mediated diseases.

Dying to protect: cell death and the control of T-cell homeostasis.

T cells play a critical role in immune responses as they specifically recognize peptide/MHC complexes with their T-cell receptors and initiate adaptive immune responses. While T cells are critical for performing appropriate effector functions and maintaining immune memory, they also can cause autoimmunity or neoplasia if misdirected or dysregulated. Thus, T cells must be tightly regulated from their development onward. Maintenance of appropriate T-cell homeostasis is essential to promote protective immunity and limit autoimmunity and neoplasia. This review will focus on the role of cell death in maintenance of T-cell homeostasis and outline novel therapeutic strategies tailored to manipulate cell death to limit T-cell survival (eg, autoimmunity and transplantation) or enhance T-cell survival (eg, vaccination and immune deficiency).

Type I interferons regulate susceptibility to inflammation-induced preterm birth.

Preterm birth (PTB) is a leading worldwide cause of morbidity and mortality in infants. Maternal inflammation induced by microbial infection is a critical predisposing factor for PTB. However, biological processes associated with competency of pathogens, including viruses, to induce PTB or sensitize for secondary bacterial infection-driven PTB are unknown. We show that pathogen/pathogen-associated molecular pattern-driven activation of type I IFN/IFN receptor (IFNAR) was sufficient to prime for systemic and uterine proinflammatory chemokine and cytokine production and induction of PTB. Similarly, treatment with recombinant type I IFNs recapitulated such effects by exacerbating proinflammatory cytokine production and reducing the dose of secondary inflammatory challenge required for induction of PTB. Inflammatory challenge-driven induction of PTB was eliminated by defects in type I IFN, TLR, or IL-6 responsiveness, whereas the sequence of type I IFN sensing by IFNAR on hematopoietic cells was essential for regulation of proinflammatory cytokine production. Importantly, we also show that type I IFN priming effects are conserved from mice to nonhuman primates and humans, and expression of both type I IFNs and proinflammatory cytokines is upregulated in human PTB. Thus, activation of the type I IFN/IFNAR axis in pregnancy primes for inflammation-driven PTB and provides an actionable biomarker and therapeutic target for mitigating PTB risk.

IL-6 and ICOS Antagonize Bim and Promote Regulatory T Cell Accrual with Age.

Regulatory T cells (Tregs), a subset of CD4(+) T cells, dramatically accumulate with age in humans and mice and contribute to age-related immune suppression. Recently, we showed that a majority of accumulating Tregs in aged mice expressed low levels of CD25, and their accrual is associated with declining levels of IL-2 in aged mice. In this study, we further investigated the origin of CD25(lo) Tregs in aged mice. First, aged Tregs had high expression of neuropilin-1 and Helios, and had a broad Vß repertoire. Next, we analyzed the gene expression profile of Tregs, naive T cells, and memory T cells in aged mice. We found that the gene expression profile of aged CD25(lo) Tregs were more related to young CD25(lo) Tregs than to either naive or memory T cells. Further, the gene expression profile of aged Tregs was consistent with recently described "effector" Tregs (eTregs). Additional analysis revealed that nearly all Tregs in aged mice were of an effector phenotype (CD44(hi)CD62L(lo)) and could be further characterized by high levels of ICOS and CD69. ICOS contributed to Treg maintenance in aged mice, because in vivo Ab blockade of ICOSL led to a loss of eTregs, and this loss was rescued in Bim-deficient mice. Further, serum levels of IL-6 increased with age and contributed to elevated expression of ICOS on aged Tregs. Finally, Treg accrual was significantly blunted in aged IL-6-deficient mice. Together, our data show a role for IL-6 in promoting eTreg accrual with age likely through maintenance of ICOS expression.

Slp-76 is a critical determinant of NK-cell mediated recognition of missing-self targets.

Absence of MHC class I expression is an important mechanism by which NK cells recognize a variety of target cells, yet the pathways underlying "missing-self" recognition, including the involvement of activating receptors, remain poorly understood. Using ethyl-N-nitrosourea mutagenesis in mice, we identified a germline mutant, designated Ace, with a marked defect in NK cell mediated recognition and elimination of "missing-self" targets. The causative mutation was linked to chromosome 11 and identified as a missense mutation (Thr428Ile) in the SH2 domain of Slp-76-a critical adapter molecule downstream of ITAM-containing surface receptors. The Slp-76 Ace mutation behaved as a hypomorphic allele-while no major defects were observed in conventional T-cell development/function, a marked defect in NK cell mediated elimination of ß2-microglobulin (ß2M) deficient target cells was observed. Further studies revealed Slp-76 to control NK-cell receptor expression and maturation; however, activation of Slp-76(ace/ace) NK cells through ITAM-containing NK-cell receptors or allogeneic/tumor target cells appeared largely unaffected. Imagestream analysis of the NK-ß2M(-/-) target cell synapse revealed a specific defect in actin recruitment to the conjugate synapse in Slp-76(ace/ace) NK cells. Overall these studies establish Slp-76 as a critical determinant of NK-cell development and NK cell mediated elimination of missing-self target cells in mice.